During development of the embryonic vertebrate brain, specification of the correct number of neurons and placement of neurons in the appropriate regions is crucial for assembling the complex network of the adult brain. We study the specification and placement of neurons in the pineal complex, the region of the brain which secretes the hormone melatonin and plays a central role in coordinating the daily and seasonal rhythms of the body. Because of the difficulty in studying human brain development, we use a model organism, the zebrafish, which allows powerful genetic and embryological experiments. Intriguingly, the pineal complex of zebrafish displays robust left-right asymmetry in its development, and influences asymmetry of nearby regions of the brain. The zebrafish pineal complex includes the large midline pineal organ, and the left-sided parapineal organ. In an effort to understand the genes and molecules that direct the formation of the pineal and parapineal organs, we have isolated mutants with an excess or dearth of cells. Novel mutants include from beyond, which causes no parapineal organ to form, and big time, which results in about twice as many pineal cells as normal. The from beyond mutation disrupts the tbx2b transcription factor, while the big time mutation has not been cloned. We also have found that floating head, a transcription factor that regulates pineal neurogenesis, does not affect development of the parapineal. We propose to use these mutations to genetically dissect the formation of the pineal and the asymmetric parapineal organ. In addition, we will screen for new mutations that affect pineal complex development. Using cell fate mapping, chimeric analysis, and epistasis experiments with these mutants, we will shed light on the genetic pathways that assign cells to a pineal or parapineal fate. Relevance: The pineal complex and its hormone melatonin regulate a wide variety of metabolic, immunological, and cardiovascular processes and can enhance the action of multiple drugs and hormones. Studying how cells of the pineal complex are formed is crucial for a better understanding of how the pineal gland, the primary source of melatonin in the body, develops and is integrated into the brain.